1. Field of the Invention.
The present invention relates to the preparation of liquid specimens by vitrification for examination of microstructure which includes morphology (size, shape, form), connectivity, topography and texture, by electron microscopy.
2. Description of the Prior Art.
Vitrification as used herein means rapidly cooling a liquid without crystallization into a highly viscous, low vapor pressure, glassy state. Vitrification is an important technique for electron microscopy of frozen hydrated biological and colloidial dispersions and aggregates.
The specimen preparation procedure prior to vitrification consists of the following steps: a specimen support plate (typically a transmission electron microscopy grid) is held by a tweezer mounted on a plunger. A drop of liquid sample to form the specimen is placed on the support plate and most of the liquid is removed by touching the liquid sample on the support plate with a blotting paper or other absorbant material to leave a very thin film of liquid on the support plate. Next, the vitrification process is carried out by allowing the specimen support plate to plunge under the force of gravity into a cooling medium (cryogen), typically melting ethane. This procedure in the prior art was done in an open laboratory atmosphere where temperature and concentration of gasses and vapors cannot be controlled.
The preparation of biological samples for electron microscopy by a vitrification technique is discussed by Adrian et al. in an article entitled "Cryo-Electron Microscopy of Viruses", Nature, Vol. 308, No. 58-54, pgs. 32-36, 1984. The value of rapidly cooling and vitrifying the samples is illustrated.
In preparing samples prior to vitrification, it has been found that when using previously established techniques, preserving the morphology, component chemical potential and connectivity, topography and texture similar to the native state of the sample is extremely difficult. Thus, specimens prepared in an uncontrolled laboratory atmosphere are subject to evaporation, which concentrates solutions and suspensions as much as three fold, and also the specimens are subject to temperature changes. These actions change the chemical potentials of the system components and can result in change of phase (for example precipitation of solids from solution) and change in microstructure.
There are instruments that have been used to attempt to vitrify liquid specimens. For example, the Reichert Scientific Instruments Division of Warner Lambert Technologies Inc. of Buffalo, N.Y. 14240 sells a model KF80 Immersion Cryofixation System. However, this system primarily maintains the cryogens that are used for vitrifying the specimen, and does not provide for preparation of the sample in a controlled atmosphere or environment prior to immersion into a cryofixation medium.
SUMMARY OF THE INVENTION
The present invention relates to preparation of samples or specimens, including aqueous solutions and suspensions, for examination by electron microscopy or by other types of microscope inspection. The present system permits preparation of specimens or samples having appreciable vapor pressure in a way that the microstructure can be examined under low pressure conditions. This is important in many technological fields including but not limited to study of colloids, surfactant dispersions, polymers, viruses, biological cells, foams and emulsions, particulate suspensions, sols, gels, ceramics, microelectronic elements and assemblies, fluid phase reaction systems, nucleation, and phase separation and catalysis. Liquids in their native state cannot be conveniently examined by electron microscope because of high vacuum in an electron microscope. Vitrification of liquid components of systems, followed by cold stage electron microscopy permits examining the microstructure of a specimen unchanged or very little change from the native microstructure of the original liquid sample. However, specimen preparation in an uncontrolled environment causes evaporation and temperature changes that may lead to artifact formation (change in morphology, connectivity, topography or texture) and phase changes. It should be noted that artifact formation may also be caused by mechanical stresses imposed during preparation of the thin sample film on the specimen support plate.
The present invention provides a way of preparing samples of all types of liquid systems, but it is especially advantageous for systems which are highly susceptible to artifact formation. Reduction of artifact formation is accomplished by controlling component chemical potentials and thus the present invention is highly advantageous for systems near a thermodynamic phase boundary, that is, particularly susceptible to phase separation. The present invention includes means which permit specimen or sample preparation at temperatures substantially different from ambient. The present invention permits extended holding of the thin film specimen in an environmental chamber, prior to vitrification, to allow reequilibration of the samples from effects of mechanical stresses induced during thinning.
The present invention comprises an environmental chamber in which the specimens or samples are prepared. The chamber atmosphere can be saturated with one or more chemical components if desired, and its temperature can be closely controlled, to avoid component chemical potential changes in the sample and reduce artifact formation and prevent phase change of the specimen. The environmental chamber has controlled means for saturating the atmosphere in the chamber with desired gases or liquid vapors, and has a temperature controlled heater and cooler for changing and controlling the chamber temperature while the specimen is being prepared. Suitable thermal sensing elements (thermistors or thermocouples for example) and a temperature controller to regulate the power to the heater or cooler are used. The temperature can be measured at several points inside the chamber if desired. Likewise, humidity or vapor saturation inside the chamber can be measured with suitable sensors, such as existing capacitive thin film sensors, and the measurements used to automatically control the environment in a desired manner.
The walls of the environmental chamber are formed to include suitable clear material so that the interior of the chamber can be viewed. The chamber has an access door which can be opened for placing a specimen holder onto a plunger shaft that is slidably mounted in the compartment in registry with a camera shutter on the lower wall of the chamber. The camera shutter is aligned with and is spaced slightly above a container of cryogenic material into which the specimen or sample is to be plunged. The lower end of the plunger shaft as shown in the accompanying drawings is adapted to hold a tweezer which in turn holds a specimen grid or support plate. When the tweezer and specimen support plate are properly positioned, the access door to the environmental chamber is manually closed, and then the atmosphere in the chamber is controlled to reach its desired state of temperature and gas and vapor content. The specimen or sample is introduced using a pipette operated through a split rubber septum to reduce changes in chamber temperature and atmosphere composition. The liquid sample is placed on the specimen support plate and is blotted to form a very thin film. Alternatively, spontaneously wetting films may be formed on the specimen support plate. The chamber atmosphere is controlled to insure that the film will not change. The specimen or sample is thus prepared completely within a chamber having a controlled environment so the specimen remains representative of the original liquid sample.
When the specimen has been prepared, a cryogenic bath, which preferrably is melting ethane or alternatively, propane or selected freons, maintained in a liquid nitrogen bath, in an insulated container is positioned below the shutter that is in the bottom wall of the environmental chamber. A double cable release with a single actuator button is used for first releasing the plunger shaft and after a selected very small time delay quickly opening the camera shutter so that the shaft and specimen support plate plunges through the shutter opening and the specimen is dropped under provided force (gravity or a spring load, for example) without any opportunity for evaporation or change in the state of specimen on the specimen support plate prior to vitrification.
A self-cocking photographic camera shutter, such as a Prontor shutter is used. This is an iris type shutter that opens quickly and can be kept closed until desired, to enable retaining environmental control and preventing the specimen from being precooled by the cold cryogen vapors that are directly below this shutter. The use of a double cable release to trip the mechanism including the plunger or shaft that plunges the specimen into the cryogen material and subsequently opens the shutter, permits precise timing by adjusting the length of the cable release pins.
The plunger shaft is held in place before plunging with a releasable pin that is moved by operation of the cable release.
The specimen or sample is then handled in a desired manner and transferred to the electron microscope for examination.